Hemophilia A affects 1:5000 males and is due to deficiencies of the coagulation factor (F) VIII. Therapy for such patients has markedly improved. One of the remaining major challenges in their care is the development of inhibitors to FVIII in 20-30% of these patients. Many of such individuals respond to immunomodulation, but a significant subset are recalcitrant to such therapy and are not fully responsive to FVIII bypass agents. Such patients often develop large joint damage and recurrent/life-threatening intracerebral bleeds. We and others have shown that FVIII ectopically expressed in platelets (pFVIII) is stored in alpha granules and released following platelet (Plt) activation. This pFVIII s at least partially protected from circulating inhibitors in murine models so that it represents a potential alternative care to FVIII bypass agents. However, we have shown that the spatial/temporal availability of pFVIII differs from plasma FVIII making it more hemostatically effective in some settings and less so in others. We have also shown that ectopic FVIII expressed in developing megakaryocytes (Megs) can be deleterious to these cells resulting in increased apoptosis during megakaryopoiesis, affecting Meg and Plt recovery post-bone marrow transplantation (BMT) in mice. We also have shown that a FVIII variant that enhances FVIII single-chain stability (FVIIIR1645H) markedly enhances pFVIII hemostatic efficacy in murine hemostasis studies. Based on our studies, we caution translating pFVIII therapy to patient care using a BMT-based strategy. We propose instead a pFVIII Plt prophylactic infusion strategy. The following three aims propose to test the efficacy of pFVIII in clinically relevant hemostatic challenges for patients with inhibitors and then develop the pFVIII Plt infusion therapy as a novel approach in the care of such patients. Specific Aim (SA) #1. Define the efficacy of pFVIII in clinically relevant models of hemostasis. pFVIII efficacy has been tested mostly in murine models that do not develop large joint bleeds or intracerebral bleeds seen in patients with inhibitors. We propose using a FVIII null rat model of spontaneous joint and intracerebral bleeds established by our group and an intracerebral photochemical injury model to test the efficacy of pFVIII in these target settings, especially with concurrent inhibitors. SA#. Optimize expression levels and pFVIII specific activity in human Megs derived from induced pluripotent stem cells (iPSCs). Using a safe harbor, adeno-associated virus site 1, insertion approach as well as a lentiviral approach, we will optimize the level activity of FVIII in Megs derived from iPSCs and characterize the injury incurred by these Megs. pFVIII levels and its in vitro hemostatic efficacy in Plts released from these Megs will also be examined. SA#3. Define the hemostatic efficacy of the pFVIII/iPSC-derived Plts. FVIII null immuno-deficient NOD/SCID/IL2R?-deficient (NSG) mice will be infused with derived Plts from Specific Aim 2, and the hemostatic efficacy of these Plts in protecting the mice will be tested in the absence and presence of inhibitors and presence of co-infused FVIII bypass agents.